Project Summary Cell-cell junctions control cell adhesion and are characterized by arrays of transmembrane adhesion proteins bound between the plasma membranes of two apposed cells. Numerous cell-cell junctions, including tight junctions, gap junctions, synapses, and others, are conserved among vertebrates and play crucial biological roles, yet despite their central importance, there are currently no satisfactory methods to investigate their detailed three-dimensional structures. Here I propose new methods for reconstituting and imaging cell-cell junctions by cryo-electron tomography (cryo-ET), which I will develop and validate with cadherin-mediated intercellular junctions. Cadherin cell adhesion proteins provide the intercellular attachments of desmosomes and adherens junctions. Cadherins of these junctions self-assemble to form dense molecular lattices dependent on specific cis (same cell) and trans (apposed cell) interactions between cadherin ectodomains. I will study the junctions produced by two types of cadherins: vascular endothelial (VE) cadherin, which forms the well-characterized endothelial junctions of blood vessels, and cadherin-11, which is normally expressed during wound healing, and for which over-expression can lead to rheumatoid arthritis. I will produce reconstituted junctions with each protein using either liposomes or large, flat lipid nanodiscs, and use cryo-ET to determine the structures of the assembled junctions. I will also continue to develop my cryo-ET pipeline that employs a novel, fiducial marker-free method for processing image data. I will supply the resulting technical cryo-ET methods and software to the scientific community, thus widening the impact of this research project. Overall, this project is expected to produce the first detailed 3D structures of endothelial junctions produced by VE-cadherin, and the first structures of a junction formed by a type II cadherin, cadherin-11. I expect this method to be generally applicable to the study of cell-cell junctions, opening possibilities for many future studies.